The goals for this proposal are to investigate toxicity and function of lentiviral vector delivered, proven gene- based HIV-1 disruption modalities in human hematopoietic stem cells (HSCs), myeloid and CD4+ T cells. It is estimated that 30-45% of AIDS patients on Highly Active Anti-Retroviral Therapy (HAART) having demonstrable virus have some form of drug resistance. Thus, the continuing emergence of drug resistant HIV-1s makes it imperative to develop therapeutics with novel mechanisms of action. We posit that by employing a combination of proven, gene-based HIV-1 disruption modalities, and the use of lentiviral vectors for delivery to CD34+ and T cells, anti-HIV-1 gene positive cells may be refractory to infection, not replicate virus efficiently, and protected cells will enrich over time. Our long-term goals are to provide basic research findings on viral and cellular gene disruption strategies that may find use for autologous T and CD34+ cell or allogenic CD34+ cell transplant sources for patients that have or are going to fail HAART. Analogous to combination drug strategies used for HAART, we are completing a lentiviral delivery vector containing multiple genes that target cellular and HIV messages and proteins, thereby disrupting viral entry, integration, viral and cellular function. Moreover, target sites have been selected that if mutations arise the resulting virus should be less fit. Our goals will be achieved by the successful completion of four highly interactive Specific Aims: 1) Complete lentiviral vectors containing multiple HIV-1 disruption genes. 2) Do anti-HIV lentiviral vectors provide protection from HIV-1 while not disrupting normal cellular function? 3) Does expression of anti-HIV genes alter human hematopoiesis and thymopoiesis? 4) Do HSCs containing anti-HIV genes give rise to myeloid and CD4+ T cells that are protected from HIV-1 challenge? When completed these studies will provide information on whether lentiviral vectors containing multiple anti- viral genes targeting HIV-1 and its cellular pathways disrupt hematopoietic function. Our findings will also provide insight into whether selected combinations of anti-viral genes confer a cell protective and selective advantage in the presence of HIV-1-infection. Lastly, it is anticipated that many of the gene disruption and lentiviral vector delivery strategies developed and validated during our studies will be applicable for other basic research and clinical uses. Project Narrative: It is estimated that 30-45% of AIDS patients on Highly Active Anti-Retroviral Therapy (HAART) having demonstrable virus have some form of drug resistance. Thus, the continuing emergence of drug resistant HIV-1s requires new therapies for treatment. Our long-term goals are to provide basic research findings on gene delivery of viral and cellular anti-HIV-1 strategies that make blood cells resistant to HIV-1 infection and reduce growth of HIV-1. These new therapies may be utilized for T cell and CD34+ cell transplant sources for patients that have or are going to fail HAART.